Effect Of Enteropathogenic E. Coli And The Mutant Strain UMD 874 Infection On Intestinal Mucosal Barrier Function And Gut Microflora

Enteropathogenic Escherichia coli (EPEC) infection is a main cause of infantile watery diarrhea. EPEC colonizes host intestinal epithelial surface and induces a characteristic histopathological alteration, referred to as the attaching/effacing (A/E) lesion. A/E lesion is characterized by intimate attachment of the bacteria to the epithelial cell membrane, accompanied by the loss of enterocyte microvilli and the formation of actin pedestals which resulted from the rearrangement of host cytoskeleton beneath adherent bacteria. A/E lesion was induced by the effector virulence proteins encoded by a pathogenicity island described as the locus of enterocyte effacement (LEE), which was delivered into host cells by a type III secretion system (TTSS). EspF is a type of EPEC secreted protein, which was to be reported to play roles in antiphagocytosis, apoptosis, tight junction breakdown, intermediate filament degradation and aquaporin redistribution. Recent study has demonstrated that EspF is essential for the decrease in TER and alteration of intestinal epithelial TJ structure in vitro.Tight junction (TJ) establishes an important barrier to prevent pathogenic microbes from entering the lamina propria. TJ regulates paracellular permeability, which forms a selective permeable barrier permitting the passive entry of nutrients, ions and water while restricting the passage of macromolecules and pathogens. The in vitro studies demonstrated that EPEC infection decreased transepithelial resistance (TER) of polarized epithelial monolayers, and disrupted tight junction (TJ) architecture and barrier function by inducing loss of TJ protein-protein interactions, redistribution of TJ proteins, dephosphorylating and dissociating occludin from intestinal epithelium.The gut microbiota has a crucial impact on a wide range of host biological processes, including metabolism of dietary nutrients, promoting proper development and homeostasis of the immune system. In addition, the microbial community residing within the intestine provides an efficient barrier against colonization by pathogenic microorganisms commonly referred to as "colonization resistance". The gut microbiota also plays a role in aiding in the development of the mucosal epithelium. In the healthy intestine, the microbiota is quite stable, whereas, imbalances of the gut microbiota has been linked to the etiology of a spectrum of human diseases, including inflammatory bowel disease (IBD), obesity and even colorectal cancer.Until recently, most of our knowledge about the effect of the A/E pathogen EPEC on tight junction functions comes from the in vitro study using epithelial cell lines in tissue culture. However, the impact of EPEC infection on tight junction in vivo and shifts in the overall structure of the intestinal microbiota during EPEC infection are poorly investigated.In the present study, we used the EPEC infected mice as an in vivo model and investigated the effect of EPEC on the localization of tight junction proteins and tight junction barrier function. And the role of EspF was determined. The composition and dynamics of the gut microbiota in response to EPEC infection was monitored by 16S rRNA PCR-DGGE. And the specific involvement of TJ membrane microdomains in EPEC invasion into host cells was determined.PART I:Molecular basis of the intestinal mucosal barrier function disruption caused by enteropathogenic E. coli infectionObjective:To investigate the effect of enteropathogenic E. coli (EPEC) infection on tight junction (TJ) structure, localization of TJ proteins in TJ membrane microdomains and intestinal epithelial barrier function.Methods:C57BL/6J mice were randomly divided into the following groups including control group, EPEC infection for 1 d,3 d and 5 d and the mutant strain UMD874 infection for 1 d,3 d and 5 d (four animals in each group). The EPEC infection mouse model was established by oral gavage with 2×10 EPEC or UMD874 suspended in 200μl of sterile PBS per day. Histological analysis of the colon tissue was carried out by routine H&E staining. TJ ultrastructure was studied by transmission electron microscopy and a molecule tracer biotin was used to examine the paracellular permeability of the colon. The localization of TJ proteins occludin, ZO-1, claudin-1,3 and 5 in the intestinal epithelium was investigated by immunofluorescence microscopy. And Western blot analysis was used to examine the distribution of TJ proteins occluding and claudin-1 in TJ membrane microdomains.Results:Our results demonstrated that TJ ultrastrcture was disrupted following EPEC infection. And the morphological changes of TJ were accompanied by increased paracellular permeability which was evidenced by the penetration of biotin into the lamina propria. Immunofluorescency analysis revealed that TJ proteins were translocated from villous membrane to the cytoplasm in intestinal epithelial cells during EPEC invasion. Western blot analysis revealed that the tight junction proteins, occludin and claudin-1 were displaced from tight junction membrane microdomains to Triton-X 100 soluble fractions after EPEC infection. Moreover, the effect of UMD874 on tight junction was less than the wild type EPEC.Conclusion:Our findings demonstrated that EPEC infection in vivo redistributed TJ proteins in TJ membrane microdomains, which led to disruption of tight junction barrier function. In addition, the EPEC secreted protein EspF play a role in the early stage during EPEC infection.PART Ⅱ:Molecular basis of the intestinal mucosal barrier functionObjective:The aim of this study was to investigate the impact of EPEC infection on host microflora, and to compare the different effect of wild type EPEC and the mutant strain.Methods:The establishment of the in vivo EPEC infection model was the same as that in part Ⅰ, except that the infection time was elongated to 21 days. The bacteria strains used in this study were the wild type EPEC 2348/69 and the mutant strain UMD874.16S rRNA PCR-DGGE was used to monitor changes in the community structure of the microbiota in the host. And the Paneth cells in the intestinal cypts were examined by routine H&E staining.Results:Analysis of the DGGE fingerprint by UPGMA method revealed that the mucosa-associated microbial ecology and the community structure of the microbiota in the feces of the host were significantly altered by EPEC infection. The microflora of the host intestine had a little recovery at 21 d postinfection. The similarity indices were 58% and 54% as compared with the control. In addition, the effect of the mutant strain on host intestinal microflora was smaller that the wild type. And the population structure of the host intestinal microflora recovered 21 d postinfection. Moreover, the amount of the Paneth cell secreted granules increased in response to EPEC infection.Conclusion:EPEC infection led to an obvious disturbance of the host intestine microflora, and it recovered gradually at the late stage. Moreover, the mutant strain had less effect than the wild type strain.PART Ⅲ:Molecular basis of the intestinal mucosal barrier function disruption caused by enteropathogenic E. coli infectionObjective:To investigate the route through which EPEC invade the host cells and the underlined molecular mechanisms.Methods:Caco-2 cells were cultured on Transwell filters to form cell monolayers, and then the cell monolayer was exposed to EPEC. The transepithelial electrical resistance was measured using an EVOM voltohmmeter. Distribution of tight junction proteins was assessed by immunoblotting and confocal imaging. Internalization of EPEC was investigated by Immunofluorescence microscopy and transmission electron microscopy.Results:EPEC attached to Caco-2 surface induced a characteristic AE lesion. EPEC infection caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of tight junction protein occludin and ZO-1 from an intercellular to cytoplasm location after EPEC invasion. Subcellular fractionation using discontinuous sucrose density gradients and Western blot analysis demonstrated a redistribution of flotillin-1, the marker protein of lipid rafts, and TJ protein occludin from TritonX-100 insoluble membrane fractions into TritonX-100 soluble fractions following exposure to EPEC. Flotillin-1 was recruited to sites of EPEC entry. Immunofluorescence microscopy and transmission electron microscopy revealed that EPEC entered host cells through tight junction.Conclusion:Our results indicated that EPEC infection caused a redistribution of tight junction proteins, leading to disruption of epithelial barrier function. EPEC colocalized with TJ protein, and EPEC entered host cells through tight junction membrane microdomains. TJ membrane microdomains played a key role in the entry of EPEC into host cells.